PPAR-gamma activators, HDAC inhibitors and their therapeutical usages

ABSTRACT

The invention relates to a composition for induction of activity of a nuclear receptor PPARγ and inhibition of HDAC in a subject in need thereof, which comprises a synergistic combination of benzoate and phenylbutyrate and/or phenylacetate in association with a pharmaceutical carrier.

BACKGROUND (a) Field

The subject matter disclosed generally relates to a novel composition ofmolecules, including benzoate (BNZ) and phenylbutyrate (PBA) thatactivates PPARγ and inhibit HDAC and describe their therapeuticalusages.

(b) Related Prior Art

Benzoate (BNZ) and phenylbutyrate (PBA) were individually studied forpossible bioactivities in reducing inflammation, cancer growth andalleviating symptoms in animal models of experimental neurologicaldisorders, namely multiple sclerosis, ALS, Huntington's disease andencephalopathy.

The clinical use of BZN and phenylacetate (PAA) in lowering plasmaammonium levels in patients with lethal hyperammonemia was established(1, 2). In certain metabolic diseases resulting from defects in ureacycle enzymes, ammonium, which cannot be converted to urea, accumulatesto a toxic level that can be lethal. A drug combination of PAA and BNZis particularly useful to treat patients with congenital errors ofmetabolism of the urea cycle enzyme, thus preventing complications suchas encephalopathy (1, 2). In fact, PAA, through mitochondrialconjugation with glutamine, results in the formation ofphenylacetylglutamine compound. Similarly, BNZ combines with glycineforming benzoylglycine (hippuric acid). These two non-toxic compounds,phenylacetyglutamine and benzoylglycine are easily eliminated in theurine. Enns et al. (2) reported the results of a 25-year clinical study,using a drug consisting of a combination of PAA and BNZ, to treatpatients with urea cycle disorders, demonstrating an overall survivalrate of 84%.

PAA and phenylbutyrate (PBA) both display important bioactivities otherthan those cited above. Chemically, PAA and PBA belong to a group ofaromatic fatty acids having a stable phenyl ring. These compounds wereproven useful in the treatment of several diseases, including, sicklecell anemia (3), amyotrophic lateral sclerosis (4), Huntington's disease(5), neuronal inflammatory conditions (6) and cancer (7). Initially, PAAwas discovered as a plant hormone that regulates cell growth (8). It hasbeen extensively studied in the past two decades as an anti-cancer agentand cellular differentiating compound in laboratory settings andclinical trials. In fact, PAA inhibits the growth of several cancer celltypes of different lineages and, in some instances, it promotes theirdifferentiation to a non-cancerous phenotype. Of interest are theeffects of PAA and PBA on gliomas and neuroblastomas, originally thoughtto be mediated by the inhibition of protein prenylation as well ascholesterol and fatty acid biosynthesis (5). Studies have demonstratedthat PAA and PBA inhibit the growth of several neoplastic cell types,including breast cancer, prostate cancer, colon cancer and thyroidcarcinoma. These anti-cancer actions of PAA and PBA have prompted theinitiation of several clinical trials since these compounds displaylittle toxicity if any (4, 5, 7).

The exact mechanisms underlying these physiological and pharmacologicaleffects of BNZ, PAA and its butyrate metabolites are not completelyknown, but regulatory effects of butyrate derivatives have involvedinhibition of histone deacetylation, which modulates chromatinconformation and regulation of nuclear receptor gene expression (9).

The present applicant PCT co-pending application published under No.WO2017/091895 in the name of Theriac Biomedicale Inc. discloses acomposition for induction of activity of a nuclear receptor PPARγ in asubject in need thereof, which comprises at least one of benzoate or asynergistic combination of benzoate and phenylacetate in associationwith a pharmaceutical carrier. There is no disclosure or suggestion of acomposition comprising PBA and BZA in this co-pending application.

The pharmacokinetic known effects of PBA and PAA are so different thatthese compounds are not used interchangeably. More precisely, PBA is amore effective anti-cancer and a more potent antitumor agent than PAA:PBA is effective against cervical cancer cells whereas PAA is not; PBAis more effective than PAA against breast cancer cells and malignant Bcells.

However to date, PBA and BNZ were never combined to show a synergisticeffect as activators of PPARγ.

SUMMARY

According to an embodiment, there is provided a composition forinduction of activity of a nuclear receptor PPARγ in a subject in needthereof, which comprises a synergistic combination of benzoate andphenylbutyrate in association with a pharmaceutical carrier.

The use of the composition, wherein the induction of activity of anuclear receptor PPARγ improves symptoms of at least one of Inflammationand pain (osteoarthritis, rheumatoid arthritis), pain, autoimmunediseases (Lupus erythematous), neurodegenerative inflammatory diseases(Multiple Sclerosis, Parkinson disease, Alzheimer, ALS, Huntington),anti-cancer, diabetes type 2, to replace PPARγ agonists in othermetabolic diseases.

According to another embodiment, there is provided use of a synergisticcombination of benzoate and phenylacetate in association with apharmaceutical carrier for inducing activity of a nuclear receptor PPARγin a subject in need thereof.

The use of this combination, wherein the inducing activity of a nuclearreceptor PPARγ improves symptoms of at least one of inflammation andpain (osteoarthritis, rheumatoid arthritis), psoriatic arthritis,juvenile arthritis, ankylosing spondylitis, gout, pain, autoimmunediseases (Lupus erythematous), neurodegenerative inflammatory diseases(Multiple Sclerosis, Parkingson disease, Alzheimer, ALS, Huntington),anti-cancer, diabetes type 2, to replace PPARγ agonists in othermetabolic diseases.

According to another embodiment, there is provided a method of inducingactivity of a nuclear receptor PPARγ in a subject in need thereof, whichcomprises administering to the subject a synergistic combination ofbenzoate and phenylacetate in association with a pharmaceutical carrier.

The inducing activity of a nuclear receptor PPARγ improves symptoms ofat least one of inflammation and pain (osteoarthritis, rheumatoidarthritis), psoriatic arthritis, juvenile arthritis, ankylosingspondylitis, gout, pain, autoimmune diseases (Lupus erythematous),neurodegenerative inflammatory diseases (Multiple Sclerosis, Parkinsondisease, Alzheimer, ALS, Huntington), anti-cancer, diabetes type 2, toreplace PPARγ agonists in other metabolic diseases.

Features and advantages of the subject matter hereof will become moreapparent in light of the following detailed description of selectedembodiments, as illustrated in the accompanying figures. As will berealized, the subject matter disclosed and claimed is capable ofmodifications in various respects, all without departing from the scopeof the claims. Accordingly, the drawings and the description are to beregarded as illustrative in nature, and not as restrictive and the fullscope of the subject matter is set forth in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present disclosure will becomeapparent from the following detailed description, taken in combinationwith the appended drawings.

FIG. 1 illustrates the time-course of carrageenan-induced acuteinflammation study design.

FIG. 2 illustrates histology examples that HIP-002 reduces thecarrageenan-induced leukocyte tissue infiltration. Acute pawinflammation was induced by intraplantar carrageenan injection asindicated in FIG. 1. Tissue leukocyte infiltration was evaluated bymicroscopy 4-hours post carrageenan injection in hematoxylin-eosinstained paw tissue sections. Photos are representative of a placebomouse injected with 0.9% NaCl (A-B), a positive control mouse injectedwith 3 mg/kg indomethacin (FIGS. 2C and 2D) and a mouse treated withHIP-002 (FIGS. 2E and 2F) consisting of a mixture of 100 mg/kg sodiumbenzoate and 10 mg/kg sodium phenylbutyrate. The same areas outlined in2A, 2C and 2E are shown at higher magnification, respectively, in 2B, 2Dand 2F. Bars=100 μm.

FIG. 3A illustrates that treatment with HIP-002 reducescarrageenan-induced leukocyte infiltration. Legend: Acute inflammationwas induced by intraplantar carrageenan injection into the right hindpaw. Tissue leukocyte infiltration was quantified under the microscopeby an expert hematologist blinded to treatment conditions.Hematoxylin-eosin stained paw tissue sections were evaluated at 4-hourspost carrageenan injection. Placebo group received 0.9% NaCl solution,the positive control group received indomethacin (3 mg/kg i.p.). Theother tested groups as shown, were a combination of benzoate 100 mg/kgof benzoate (BZN) and 10 mg/kg of either PBA or PAA. Total leukocytes(white blood cells) per tissue area were quantified. All data arepresented as mean±SEM. P values versus placebo in one-way ANOVA followedby Holm-Sidak are depicted on the graph by (**) indicating p<0.01.Number of mice per group is 10 but, on rare occasions, the evaluatorscored only 5 of the animals.

FIG. 3B illustrates that HIP-002 reduces carrageenan-induced paw edema.Acute inflammation was induced by intraplantar carrageenan injectioninto the right hind paw. Paw edema was determined by measuring pawvolume before and 4-hours after carrageenan injection. The placebocontrol group received 0.9% NaCl solution (i.p.) and the positivecontrol group received indomethacin (3 mg/kg). Treatments were given byintraplantar injection (together with carrageenan) and by i.p. injection(90 min after carrageenan). BZN+PBA doses were 100 mg/kg BZN and 10mg/kg of PBA (100/10). The far right group received BZN 100 mg/kg andPAA 10 mg/kg. FIG. 3C illustrates that HIP-002 as compared to control(placebo) can significantly reduce pain associated allodynia and more sothan the NSAID positive control (indomethacin) as tested by classicmethods (10, 11). All data are presented as mean±SEM. (*) P<0.05 versusplacebo in one-way ANOVA followed by Holm-Sidak. Number of mice pergroup is 10. Briefly, FIGS. 3A, 3B and 3C show that HIP-002 reducesinflammatory cell infiltration, edema and allodynia.

FIG. 4A illustrates the effects of HIP-002 on in vitro assays of PPARγactivities. Human embryonic kidney (HEK) 293 cells were transfected witha luciferase reporter gene construct under the control of a Gal4-DNAbinding upstream-activating sequence (UAStkLuc) in the presence of anexpression plasmid encoding Gal4 DNA-binding domain fusion to humanPPARγ (12). In this assay, luciferase activity is a direct measurementof PPARγ activation.

FIG. 4B illustrates the effects of HIP-002 on in vitro assays of HDACactivities. The detection of HDAC activity is based on a two-stepenzymatic reaction. i.e., deacetylation of a lysine residue bound to afluorescent group, followed by the cleavage of the deacetylatedsubstrate and release of the free, highly fluorescent group. Themeasured fluorescence is directly proportional to the deacetylationactivity of the sample. Data represent HDAC activity in response toincreasing concentrations of HIP-002. Curves were fitted using athree-parameter non-linear fit (inhibitor-response) in GraphPad Prism®7.02.

FIG. 5 illustrates an example of histopathology in placebo and HIP-002treated rats Paraffin embedded tissue sections were obtained aftersurgical induction of osteoarthritis followed by treatment with placebo(top) or HIP-002 high dose qd (bottom). Histological sections werestained with safranin-O, a special histochemical stain for visualizationof cartilage (tissue in red). Note damaged cartilage at tibia/femurinterface in placebo treated rat, unlike the HIP-002 treated one. Also,note differences in bone eburnation and bone marrow fibrosis. 1:cartilage damage, 2: Fibrosis in bone marrow, 3: bone eburnation.

FIG. 6 is a cartoon that illustrates the proposed mechanisms of actionfor HIP-002 in the OA knee tissue, based on the pre-clinical data.

FIGS. 7A-7B, 8A-8C and 9A-9B illustrate that HIP-002 reduces pain andthe cytokine levels in the serum of rats with surgically inducedosteoarthritis (OA). FIG. 7C shows the effect of HIP-002 on the level ofsubstance Pin the spinal cord extracts, as assayed by HPLC-MS.Significant P values (<0.05) after one-way ANOVA are denoted, number ofrats per group is denoted in parentheses. Animals were treated dailywith positive control (5 mg/kg of the NSAID, carprofen po and 30 mg/kgof pregabalin sc) or HIP-002 (38 mg/kg, sc), once or twice (2×) daily.In the experiments shown, a low dose of HIP (25% of the high dose) wasalso tested.

Blood samples were withdrawn to assess inflammatory cytokine levels at1, 7 and 22 days after beginning of treatment processed for analysis ina multiplex magnetic-bead immunoassay system and reported as pg or ng/mLof serum. Data are presented as mean±SEM. Parentheses denote the minimalnumber of rats per group. Significant P values of HIP-002 versus placeboafter one-way ANOVA are denoted on the graph.

FIGS. 8 and 9 show that HIP-002, high dose, qd, significantly reducedIL-1 beta, TIMP-1, IL-18, VEGF and ICAM-1/CD54. In addition,L-Selectin/CD62L was also significantly inhibited by approximately 40%(not shown), (P<0.01) when compared with placebo. Other tested cytokineswere found to be below the detection level of the assay system.

Statistical significance is indicated, as compared to placebo. At least10 animals per group were tested. Note significant inhibitory effects byHIP-002, unlike the positive control.

FIG. 10 illustrates proposed molecular mechanism of HIP-002 action atthe genomic level.

DETAILED DESCRIPTION

The combination of benzoate and phenylbutyrate induce the activity ofthe nuclear receptor PPARγ. This transcription factor is a member of thesteroid receptor superfamily and master regulator of lipid metabolism,inflammation and a key target for insulin-regulating drugs.

Furthermore, the usefulness of concomitant treatment of benzoate andphenylbutyrate in the treatment of osteoarthritis is demonstrated in arodent model of osteoarthritis.

The present invention will be more readily understood by referring tothe following examples which are given to illustrate the inventionrather than to limit its scope.

Example 1

Indications for the Mixture of PBA & BZN in Humans and Animals

-   -   Inflammation and pain including but not limited to        osteoarthritis, rheumatoid arthritis, atherosclerosis,        periodontitis, hay fever    -   Psoriatic arthritis    -   Juvenile arthritis    -   Ankylosing spondylitis    -   Gout    -   Pain    -   Autoimmune diseases including but not limited to Addison's        disease, Agammaglobulinemia, Alopecia areata, Amyloidosis,        Ankylosing spondylitis, Anti-GBM/Anti-TBM nephritis,        Antiphospholipid syndrome (APS), Autoimmune hepatitis,        Autoimmune inner ear disease (AIED), Axonal & neuronal        neuropathy (AMAN), Behcet's disease, Bullous pemphigoid,        Castleman disease (CD), Celiac disease, Chagas disease, Chronic        inflammatory demyelinating polyneuropathy (CIDP), Chronic        recurrent multifocal osteomyelitis (CRMO), Churg-Strauss,        Cicatricial pemphigoid/benign mucosal pemphigoid, Cogan's        syndrome, Cold agglutinin disease, Congenital heart block,        Coxsackie myocarditis, CREST syndrome, Crohn's disease,        Dermatitis herpetiformis, Dermatomyositis, Devic's disease        (neuromyelitis optica), Discoid lupus, Dressler's syndrome,        Endometriosis, Eosinophilic esophagitis (EoE), Eosinophilic        fasciitis, Erythema nodosum, Essential mixed cryoglobulinemia,        Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell        arteritis (temporal arteritis), Giant cell myocarditis,        Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with        Polyangiitis, Graves' disease, Guillain-Barre syndrome,        Hashimoto's thyroiditis, Hemolytic anemia, Henoch-Schonlein        purpura (HSP), Herpes gestationis or pemphigoid gestationis        (PG), Hypogammalglobulinemia, Huntington disease, IgA        Nephropathy, IgG4-related sclerosing disease, Inclusion body        myositis (IBM), Interstitial cystitis (IC), Juvenile arthritis,        Juvenile diabetes (Type 1 diabetes), Juvenile myositis (JM),        Kawasaki disease, Lambert-Eaton syndrome, Leukocytoclastic        vasculitis, Lichen planus, Lichen sclerosus, Ligneous        conjunctivitis, Linear IgA disease (LAD), Lupus, Lupus        erythematous, Lyme disease chronic, Meniere's disease,        Microscopic polyangiitis (MPA), Mixed connective tissue disease        (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple        sclerosis (MS), Myasthenia gravis, Myositis, Narcolepsy,        Neuromyelitis optica, Neutropenia, Ocular cicatricial        pemphigoid, Optic neuritis, Palindromic rheumatism (PR), PANDAS        (Pediatric Autoimmune Neuropsychiatric Disorders Associated with        Streptococcus), Paraneoplastic cerebellar degeneration (PCD),        Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg        syndrome, Pars planitis (peripheral uveitis), Parsonnage-Turner        syndrome, Pemphigus, Peripheral neuropathy, Perivenous        encephalomyelitis, Pernicious anemia (PA), POEMS syndrome        (polyneuropathy, organomegaly, endocrinopathy, monoclonal        gammopathy, skin changes), Polyarteritis nodosa, Polymyalgia        rheumatica, Polymyositis, Postmyocardial infarction syndrome,        Postpericardiotomy syndrome, Primary biliary cirrhosis, Primary        sclerosing cholangitis, Progesterone dermatitis, Psoriasis,        Psoriatic arthritis, Pure red cell aplasia (PRCA), Pyoderma        gangrenosum, Raynaud's phenomenon, Reactive Arthritis, Reflex        sympathetic dystrophy, Reiter's syndrome, Relapsing        polychondritis, Restless legs syndrome (RLS), Retroperitoneal        fibrosis, Rheumatic fever, Rheumatoid arthritis (RA),        Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's        syndrome, Sperm & testicular autoimmunity, Stiff person syndrome        (SPS), Subacute bacterial endocarditis (SBE), Susac's syndrome,        Sympathetic ophthalmia (SO), Takayasu's arteritis, Temporal        arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP),        Tolosa-Hunt syndrome (THS), Transverse myelitis, Type 1        diabetes, Ulcerative colitis (UC), Undifferentiated connective        tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo, Wegener's        granulomatosis (now termed Granulomatosis with Polyangiitis        (GPA)    -   Neurodegenerative inflammatory diseases including but not        limited to Multiple Sclerosis, Parkinson disease, Alzheimer, ALS        or Lou Gehrig's disease, Huntington    -   Cancer    -   Diabetes type 2    -   To Replace PPAR gamma agonists in other metabolic diseases    -   To regulate gene expression by boosting histone deacetylase        inhibition.

Example 2 Anti-Inflammatory Actions of PBA and/or PAA in Combinationwith BZN

To test such a hypothesis, the anti-inflammatory effects of combined BZNand PBA and/or PAA were assessed in two inflammation models: a mousemodel of carrageenan-induced acute inflammation (Morris C J., MethodsMol Biol 2003; 225:115-21) and a rat model of osteoarthritis (OA) (10,11) Bove S E et al. Osteoarthritis Cartilage 2006; 14(10):1041-8).

These models were selected because they are well-established, reliableand reproducible rodent models that allow the evaluation of pain andinflammation. Briefly, in the carrageenan-induced acute inflammationmodel, a rapid inflammatory response is triggered by the injection of asmall volume of a solution containing carrageenan in the paw of adultmice. Paw edema, tissue leukocyte infiltration and tactile allodyniausually develop within few minutes and last several hours. Afterinduction of the lesion (carrageenan or OA), animals were tested fordrug response. All experiments included a placebo group injected withphysiological NaCl 0.9% solution, a positive control group receivingindomethacin (carrageenan-model) or pregabalin+carprofen (in the OAmodel) and one or more group receiving test doses of HIP-002 (acomposition consisting of a mixture of PBA and BZN or PAA+BZN).

Drugs for the positive control groups were chosen with the followingrationale: Indomethacin is a fast-acting non-steroidal anti-inflammatorydrug (NSAID) commonly used to treat inflammation and was used at itsminimally effective dose. The dose range of HIP-002 was selected basedon the effective concentration (EC50) obtained with experiments in vitroor in vivo pilot experiments.

Different experiments were used to evaluate diverse aspects ofinflammation, measurements of paw edema and leukocyte tissueinfiltration were done in the carrageenan test to assess acuteinflammation.

Results

Carrageenan-Induced Acute Inflammation

In this study (UOM-01-R1-Carr), acute inflammation was induced in Swissmice by intraplantar (i. plant.) injection of 20 μL 1% carrageenan (N=10or 11 mice per group). Ninety minutes after carrageenan, mice weretreated with either placebo (0.9% NaCl solution), indomethacin (3 mg/kg)or our test drug, code named HIP-002 whose concentration were (100+10mg/kg of BZN+PBA respectively). In one group, PAA was used instead ofPBA.

Test articles, including placebo, were also injected intraplantarly (10μL) at the same time of carrageenan. The study schedule assessment isdepicted in FIG. 1, doses, route and results are summarized in Table 1below.

TABLE 1 Summary of effects of HIP-002 on Carrageenan-Induced AcuteInflammation Doses and Administration Route of (BZN + PBA) Method ofNoteworthy Experiment and BZN + PAA Tests Analysis ObservationsCarrageenan- Groups Leukocyte Quantification Reduced number of inducedacute Description: Tissue of infiltrated immune cells in theinflammation saline infiltration immune cells in paw of positive controlSwiss mouse i.p.: 1 mL/kg Immune H-E stained groups (10 or 11 i.plant.10 μL cell histology BZN + PBA ↓ mice/group) 100BZN + either migrationsections of leukocyte infiltration PBA or PAA 10 paws mg/kg i.p. andi.plant. Paw Paw volume Reduced indomethacin edema measured in acarrageenan-induced i.p.: 3 mg/kg Swelling plethysmometer edema in BZN +PBA ibid i.plant. response treated group when & pain compared toplacebo. ↓ paw swelling Abbreviations: “i.p.” means intraperitoneal and“iplant.” means intraplantar

As part of its biological action, PBA and PAA is known to be a histonedeacetylase inhibitor (HDACi). The HDACi action induces conformationalchanges in chromatin, thereby regulating the overall gene expression oftarget cells (Nor et al. Mol Neurobiol 2013, 48(3):533-543). Wediscovered that by combining benzoate compounds with PBA and/or PAA, theHDACi activity is enhanced as compared to placebo or appropriatepositive control. We tested HIP-002 or PBA alone for HDACi in vitrousing normal, primary cultured (chondrocytes, osteoblasts) and cancercell lines (breast cancer, prostate, bladder, and glioblastoma) atconcentrations ranging from 1-10 mM. As positive control,trichostatin-A, a known HDACi, was used (at 100-500 nM doses). We alsotested for HDACi, the paw tissue extract homogenates (prepared from thecarrageenan or OA rodent experiments described elsewhere in this patentapplication). The HDACi activity was measured using a classic assaymethod (such as described by Sigma-Aldrich case #CS1010). We found thatthe combination of PBA (or PAA) with BZN is significantly more potentthan PBA alone. Furthermore, PBA alone or in combination is more potentthan PAA.

To test either the effect of HDAC inhibition by PBA (or PAA) or thestimulation of PPAR γ, we tested HIP-002 to induce PPARγ transcriptionalactivity using a cellular one-hybrid luciferase reporter gene assay aspreviously described (12). Human embryonic kidney 293 cells weretransfected with a luciferase reporter gene construct under the controlof a Gal4-DNA binding upstream-activating sequence (UAStkLuc) in thepresence of an expression plasmid encoding Gal4 DNA-binding domainfusion to human PPARγ. A significant and dose-dependent increase inPPARγ activity was observed upon administration of HIP-002

Results are illustrated in FIG. 4.

Example 3 Pathology Test

The overall objective of the study was to compare the efficacy of thetest item, Compound HIP-002, following either a surgically inducedosteoarthritis in female Sprague-Dawley rats and further to compare thewithdrawal thresholds using the Von Frey and Paw Withdrawal tests.

The specific objective of the Pathology sub-study was to examine thehistopathological differences between the experimental groups,particularly with respect to the extent of inflammation and cytology ofthe hyaline layer and other cells of the knee joint.

Experimental Design

The test item, Molecule HIP-002 is described in this application. Thepositive control comprises of Pregabalin and Carprofen. The placebo(control) item was 0.9% Physiological saline USP.

The test, positive, and placebo (control)/vehicle items wereadministered to groups of rats daily as follows by oral (Gavage) (orsubcutaneous (SC) and Carprofen was administered by SC injection(administration was one after the other). Administration was for aduration of 39 or 40 consecutive days as described.

Animals were euthanized either on Day 40 or 41 upon completion of thedosing periods and following an overnight period without food. Theseanimals were anesthetized with Isoflurane followed by exsanguination.Gross pathology consisted of an external examination, includingidentification of all clinically-recorded lesions, as well as a detailedinternal examination. Pre-treatment conditioned knee samples wereretained from all animals, processed according to the study plan andexamined histopathologically.

Results and Discussion

Mortality

There were no macro or microscopic findings that could be confirmed tobe test item-related. Dark discoloration of the mandibular andmediastinal lymph nodes was regarded as procedure-related finding. Therewere no other findings seen at necropsy of this female.

Microscopically, mild diffuse chronic inflammation of the joint capsuleand mild granular basophilic material with cell debris/eosinophilicmaterial in the joint cavity was observed and considered to be caused bythe pre-treatment procedure. These findings confirmed that thepre-treatment procedure was able to cause inflammatory changes in thejoint.

Terminal Animals (Main)

Macroscopic Findings

Crust on the right knee was seen in one Group 1 animal.

All other findings were considered to be incidental orprocedure-related.

Microscopic Findings

In general, the same findings of the right knee consisting of diffusechronic inflammation of the joint capsule and granular basophilicmaterial in the joint cavity were observed in both group designationsbut with a higher incidence and severity of these findings in the kneeinjection osteoarthritic-induced method designated groups, when comparedwith the surgical osteoarthritic-induced method designated groups.

Minimal to mild diffuse chronic inflammation of the joint capsule andminimal granular basophilic material with cell debris/eosinophilicmaterial in the joint cavity were observed in the animals of all threegroups where osteoarthritis was induced by surgical method. Summary ofright knee microscopic findings are presented in

The highest severity of the findings was seen in Placebo (Control)group, where no positive treatment and/or treatment by HIP-002 wasperformed. Severity of the diffuse chronic inflammation was lower in theMolecule HIP-002 group (7 minimal and 3 mild) when compared withPositive Control group (4 minimal and 6 mild) and Placebo Control group(3 minimal and 7 mild).

Knee Injection Method Group Designation

Minimal to moderate diffuse chronic inflammation of the joint capsuleand minimal to mild granular basophilic material with or without celldebris/eosinophilic material in the joint cavity were observed inanimals of both groups where osteoarthritis was induced. While theincidence and severity of the granular basophilic material in the jointcavity was comparable in both groups, severity of diffuse chronicinflammation of the joint capsule was slightly decreased in the grouptreated by HIP-002 when compared with the Placebo Control group.

CONCLUSION

Subcutaneous administration of HIP-002 after surgically inducedosteoarthritis (section through the cruciate ligament and tear of medialmeniscus) was associated with a lower incidence of diffuse chronicinflammation of the joint capsule, when compared with Positive Controlanimals treated with Pregabalin (orally) and Carprofen (subcutaneously)and with untreated animals (Placebo Control).

Furthermore, HIP-002 administration was associated with a lowerincidence of diffuse chronic inflammation of the joint capsule, whencompared with untreated animals (Placebo Control).

Histopathology:

At day 36 animals were sacrificed and had the right knee dissected,imbedded in paraffin and sectioned for histopathology. Histologicalsections were stained with safranin-O, a special histochemical stain forvisualization of cartilage.

In FIG. 5 histological sections were stained with safranin-O, a specialhistochemical stain for visualization of cartilage (tissue in red).Sections were obtained after surgical induction of osteoarthritisfollowed by treatment with placebo (top) or HIP-002 (bottom). Notedamaged cartilage at tibia/femur interface in placebo treated rat,unlike the HIP-002 treated one. Also, note differences in boneeburnation and bone marrow fibrosis. 1: cartilage damage, 2: Fibrosis inbone marrow, 3: bone eburnation, 4: preserved cartilage, 5: preservedbone.

In FIG. 6, after induction of OA by an initial lesion (top left panel),inflammatory cytokines and substance P are released, respectively, byimmune cells and sensory neurons, to mediate inflammation and pain.HIP-002 acts by reducing cytokines and substance P levels, thuspreventing further damage caused by chronic inflammation of the injury.Following treatment with HIP-002 (top right panel) inflammation and painare reduced and the cartilage layer is repaired. An untreated lesion(bottom panel), however, leads to inflamed and infiltrated synovium,necrotic cartilage, death of chondrocytes, development of neuropathicpain and severe impairment of the articulation.

In the rat OA pain and inflammation model described above, OA is inducedby surgery (destabilization of the medial meniscus and section of thecruciate ligament). Beginning either 3 or 8 days after surgery, animalswere treated with daily administration of placebo (saline), positivecontrol (30 mg/kg pregabalin administered orally [po]+5 mg/kg carprofenadministered subcutaneously [sc]), or HIP-002. Various doses of HIP-002were tested in a factorial study. The concentrations of 100 mg/Kg sc)were later found to be optimal. At various times up to 56 days aftersurgery, rats were tested for their pain response. Tactile allodynia wasassessed using the von Frey filament paw withdrawal threshold test (FIG.7A). Ongoing/spontaneous pain (FIG. 7B) was assessed using the staticweight-bearing test. Two other tests were also done, assessments ofjoint pain by the classical pressure application method (not shown).

Treatment with HIP-002 provided significant pain relief in terms oftactile allodynia (7A) and ongoing/spontaneous pain (7B). For both painindicators, HIP-002 was at least as effective as the positive controland in some types of experiments (static weight bearing and PAM) it wassuperior. Decrease in pain was also demonstrated by neuropeptidemeasurements in spinal cord extracts from rats following HIP-002treatment. FIG. 7C shows such an example of a significant decrease insubstance P, a known biomarker for pain.

Example 4 Cytokine Tests

Reduction of Inflammation by HIP-002 is also demonstrated by asignificantly reduced level of proinflammatory cytokines and chemokines.In the rat OA model discussed above, HIP-002 was superior to the classic(veterinary use) NSAID (non-steroidal anti-inflammatory drug) carprofenin reducing inflammation and pain, as well as improving functionalityand tissue regeneration, as discussed above. One of the most remarkableresults was the reduction of serum cytokine and chemokine biomarkers(FIGS. 8 and 9). The reduction was generally in the order of 50%(P<0.001) for most tested cytokines and it was rapid (24 hrs) andsustained, till the end of the experiment (53 days post dosing).

Of note is the marked decrease in IL-1beta and IL-18, two importantcytokines in the IL-1 family. Cytokine reduction was fast, and sustainedthroughout the treatment period. This significant reduction by HIP-002contrasts with the lack of effect on placebo and on the comparatorNSAID, P<0.001, (see FIGS. 8 and 9). Equally important was thesignificant decrease of leukocyte infiltrates in tissue sections ofosteoarthritic knee joints.

VEGF, IL-1 and ICAM-1 and several others were significantly inhibited byHIP-002 (see FIGS. 8 and 9); these cytokines, among others, regulateangiogenesis, a phenomenon crucial for the proliferation of vascularendothelial cells in several diseases including cancer and the morerecent COVID-19. Angiogenesis is thought to play a key role in thepathobiology of COVID-19. Lung failure and tissue damage is due to thecytokine storm which causes lethality in these COVID-19 patients.Therefore by reducing VEGF, for example, HIP-002 may reduce angiogenesisand reduce the cytokine storm and improve patient survival. Recent dataindicate that vascular angiogenesis distinguished the pulmonarypathobiology of COVID-19 from that of equally severe influenza virusinfection (12)(Ackermann et al. New Engl. J. Med. May 21, 2020).

Exaggerated production of a multitude of cytokines and chemokines, forexample, are part of the acute inflammation often accompanied by anaggressive immune response seen following viral infection, particularlywith SARS-Cov-2. Inadequate control of the anti-inflammatory response,albeit multifactorial leads to a condition described as the “cytokinestorm” (13, 14). In some patients, the exaggerated cytokine stormstimulates additional cytokines/chemokines by macrophages and virallyinfected dendritic cells which attracts more inflammatory cells thatmigrate to the sites of inflammation and release additionalchemokines/cytokines to amplify the cytokine storm. Eventually the stormcan become overwhelming resulting in severe tissue injury and ultimatelyin sepsis and multi-organ failures which is fatal, especially insubjects with comorbidities.

CONCLUSION: Since HIP-002 strongly inhibits several cytokines thatregulate angiogenesis and inflammation, it offers important therapeuticavenues for COVID-19. Reducing the cytokine storm and angiogenesis isimportant for overcoming COVID-19 lethality.

Proposed Molecular Mechanism of Action

The exact mechanisms underlying the physiological and pharmacologicaleffects of sodium benzoate (BZN), and phenylbutyrate (PBA) and/orphenylacetylate is shown below. The key features suggest a directinhibition on HDAC (like other histone modifying agents) and activationPPARγ gene regulation (in a manner similar to steroidal hormones).

In FIG. 10, HIP-002 is made of two molecular entities, which target twoseparate but complementary pathways in the cell nucleus. (1) Inhibitionof Histone Deacetylase (HDAC), thereby regulating chromatin remodeling;and (2) activation of PPAR, resulting in interaction of PPARγ with thetranscription complex and allowing expression of key cellular proteinsthat ultimately modulate physiological responses.

While preferred embodiments have been described above and illustrated inthe accompanying drawings, it will be evident to those skilled in theart that modifications may be made without departing from thisdisclosure. Such modifications are considered as possible variantscomprised in the scope of the disclosure.

REFERENCE LIST

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The invention claimed is:
 1. A pharmaceutical composition for inductionof activity to improve disease symptoms by regulating a gene expressionusing a combination of histone deacetylase inhibitors (HDAC) withstimulators of peroxisome proliferator-activated receptors (PPAR) inmultiple target cells, comprising: a) a PPARy agonists which stimulatenuclear peroxisome proliferator-activated receptors PPAR, particularlyPPARy, that improves symptoms of at least one of inflammation, pain,autoimmune diseases, neurodegenerative inflammatory diseases, cancer anddiabetes type 2 in multiple target cells, b) a molecules that havehistone deacetylase (HDAC) inhibitory activity which possessanti-proliferative, anti-inflammatory and anti-pain actions in a subjectin need thereof, c) a synergistic combination of a benzoate and aphenylbutyrate and/or a phenylacetate in association with apharmaceutical carrier; wherein the benzoate is present between 10 to250 mg/kg of benzoate and the phenylbutyrate and/or the phenylacetate ispresent between 0.1 to 1000 mg/kg, d) administering to a subject in needthereof an effective amount of the pharmaceutical composition forinduction of activity to improve disease symptoms by stimulating thePPARy and the molecules and the synergistic combinations.
 2. Thepharmaceutical composition of claim 1 wherein the pharmaceutical carrieris chosen from water, physiological saline, or another suitablesolution.
 3. The pharmaceutical composition of claim 2, wherein saidinduction of activity of the receptor PPARy improves symptoms of atleast one of inflammation, pain, autoimmune diseases, neurodegenerativeinflammatory diseases, cancer and diabetes type 2 to replace PPARyagonists.
 4. The pharmaceutical composition of claim 3, wherein theinflammation is chosen from at least one: osteoarthritis, rheumatoidarthritis, atherosclerosis, periodontitis, hay fever; autoimmunediseases is chosen from Addison's disease, Agammaglobulinemia, Alopeciaareata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome (APS), Autoimmune hepatitis,Autoimmune inner ear disease (AIED), Axonal & neuronal neuropathy(AMAN), Behcet's disease, Bullous pemphigoid, Castleman disease (CD),Celiac disease, Chagas disease, Chronic inflammatory demyelinatingpolyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis(CRMO), Churg-Strauss, Cicatricial pemphigoid/benign mucosal pemphigoid,Cogan's syndrome, Cold agglutinin disease, Congenital heart block,Coxsackie myocarditis, CREST syndrome, Crohn's disease, Dermatitisherpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica),Discoid lupus, Dressier's syndrome, Endometriosis, Eosinophilicesophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essentialmixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosingalveolitis, Giant cell arteritis (temporal arteritis), Giant cellmyocarditis, Glomerulonephritis, Goodpasture's syndrome, Graves'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolyticanemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoidgestationis (PG), Hypogammalglobulinemia, Huntington disease, IgANephropathy, IgG4-related sclerosing disease, Inclusion body myositis(IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes(Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus, Lupuserythematous, Lyme disease chronic, Meniere's disease,Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis (MS),Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica,Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromicrheumatism (PR), PANDAS (Pediatric Autoimmune Neuropsychiatric DisordersAssociated with Streptococcus), Paraneoplastic cerebellar degeneration(PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Rombergsyndrome, Pars planitis (peripheral uveitis), Parsonnage-Turnersyndrome, Pemphigus, Peripheral neuropathy, Perivenousencephalomyelitis, Pernicious anemia (PA), POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,skin changes), Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cellaplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, ReactiveArthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsingpolychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis,Rheumatic fever, Rheumatoid arthritis (RA), Sarcoidosis, Schmidtsyndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicularautoimmunity, Stiff person syndrome (SPS), Subacute bacterialendocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transversemyelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiatedconnective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo,Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis(GPA)); and neurodegenerative inflammatory diseases is chosen fromMultiple Sclerosis, Parkinson disease, Alzheimer, ALS or Lou Gehrig'sdisease.
 5. The pharmaceutical composition of claim 3, wherein theinflammation is caused by at least one: SARS-CoV-2 infection, COVID-19,or a virus infection.
 6. The pharmaceutical composition of claim 1includes use of a physiological amount of benzoate substantially at thesame time as a physiological amount of phenylbutyrate for inducingactivity of the receptor PPARy in the subject in need thereof, whereinbenzoate and phenylbutyrate and/or phenylacetate are each in associationwith the pharmaceutical carrier.
 7. The pharmaceutical composition ofclaim 6, wherein said inducing activity of the receptor PPARy improvessymptoms of at least one of inflammation, pain, autoimmune diseases,neurodegenerative inflammatory diseases, cancer and diabetes type 2 toreplace PPARy agonists.
 8. The pharmaceutical composition of claim 7,wherein the inflammation is chosen from osteoarthritis, rheumatoidarthritis, atherosclerosis, periodontitis, hay fever; autoimmunediseases is chosen from Addison's disease, Agammaglobulinemia, Alopeciaareata, Amyloidosis, Ankylosing spondylitis, Anti-GBM/Anti-TBMnephritis, Antiphospholipid syndrome (APS), Autoimmune hepatitis,Autoimmune inner ear disease (AIED), Axonal & neuronal neuropathy(AMAN), Behcet's disease, Bullous pemphigoid, Castleman disease (CD),Celiac disease, Chagas disease, Chronic inflammatory demyelinatingpolyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis(CRMO), Churg-Strauss, Cicatricial pemphigoid/benign mucosal pemphigoid,Cogan's syndrome, Cold agglutinin disease, Congenital heart block,Coxsackie myocarditis, CREST syndrome, Crohn's disease, Dermatitisherpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica),Discoid lupus, Dressier's syndrome, Endometriosis, Eosinophilicesophagitis (EoE), Eosinophilic fasciitis, Erythema nodosum, Essentialmixed cryoglobulinemia, Evans syndrome, Fibromyalgia, Fibrosingalveolitis, Giant cell arteritis (temporal arteritis), Giant cellmyocarditis, Glomerulonephritis, Goodpasture's syndrome, Graves'disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, Hemolyticanemia, Henoch-Schonlein purpura (HSP), Herpes gestationis or pemphigoidgestationis (PG), Hypogammalglobulinemia, Huntington disease, IgANephropathy, IgG4-related sclerosing disease, Inclusion body myositis(IBM), Interstitial cystitis (IC), Juvenile arthritis, Juvenile diabetes(Type 1 diabetes), Juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus,Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD),Lupus, Lupus erythematous, Lyme disease chronic, Meniere's disease,Microscopic polyangiitis (MPA), Mixed connective tissue disease (MCTD),Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis (MS),Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica,Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromicrheumatism (PR), PANDAS (Pediatric Autoimmune Neuropsychiatric DisordersAssociated with Streptococcus), Paraneoplastic cerebellar degeneration(PCD), Paroxysmal nocturnal hemoglobinuria (PNH), Parry Rombergsyndrome, Pars planitis (peripheral uveitis), Parsonnage-Turnersyndrome, Pemphigus, Peripheral neuropathy, Perivenousencephalomyelitis, Pernicious anemia (PA), POEMS syndrome(polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy,skin changes), Polyarteritis nodosa, Polymyalgia rheumatica,Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Primary biliary cirrhosis, Primary sclerosing cholangitis,Progesterone dermatitis, Psoriasis, Psoriatic arthritis, Pure red cellaplasia (PRCA), Pyoderma gangrenosum, Raynaud's phenomenon, ReactiveArthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsingpolychondritis, Restless legs syndrome (RLS), Retroperitoneal fibrosis,Rheumatic fever, Rheumatoid arthritis (RA), Sarcoidosis, Schmidtsyndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicularautoimmunity, Stiff person syndrome (SPS), Subacute bacterialendocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia (SO),Takayasu's arteritis, Temporal arteritis/Giant cell arteritis,Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), Transversemyelitis, Type 1 diabetes, Ulcerative colitis (UC), Undifferentiatedconnective tissue disease (UCTD), Uveitis, Vasculitis, Vitiligo,Wegener's granulomatosis (now termed Granulomatosis with Polyangiitis(GPA)); and neurodegenerative inflammatory diseases is chosen fromMultiple Sclerosis, Parkinson disease, Alzheimer, ALS or Lou Gehrig'sdisease.